An association study of ABCG2 rs2231142 on the concentrations of allopurinol and its metabolites.

ABCG2 is a gene that codes for the human breast cancer resistance protein (BCRP). It is established that rs2231142 G>T, a single nucleotide polymorphism of the ABCG2 gene, is associated with gout and poor response to allopurinol, a uric acid-lowering agent used to treat this condition. It has also been suggested that oxypurinol, the primary active metabolite of allopurinol, is a substrate of the BCRP. We thus hypothesized that carrying the rs2231142 variant would be associated with decreased oxypurinol concentrations, which would explain the lower reduction in uric acid. We performed a cross-sectional study to investigate the association between the ABCG2 rs2231142 variant and oxypurinol, allopurinol, and allopurinol riboside concentrations in 459 participants from the Montreal Heart Institute Hospital Cohort. Age, sex, weight, use of diuretics, and estimated glomerular filtration rate were all significantly associated with oxypurinol plasma concentration. No association was found between rs2231142 and oxypurinol, allopurinol and allopurinol riboside plasma concentrations. Rs2231142 was not significantly associated with daily allopurinol dose in the overall population, but an association was observed in men, with T carriers receiving higher doses. Our results do not support a major role of ABCG2 in the pharmacokinetics of allopurinol or its metabolites. The underlying mechanism of the association between rs2231142 and allopurinol efficacy requires further investigation.

Thiopurine pharmacogenomics and pregnancy in inflammatory bowel disease.

The thiopurine drugs azathioprine and 6-mercaptopurine are widely used for the maintenance of clinical remission in steroid-dependent inflammatory bowel disease (IBD). Thiopurines are recommended to be continued throughout pregnancy in IBD patients, but conclusive safety data in pregnant patients remain still insufficient. On the other hand, a strong association between a genetic variant of nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15 p.Arg139Cys) and thiopurine-induced myelotoxicity has been identified. Pharmacokinetic studies have revealed that thiopurine metabolism is altered in pregnant IBD patients and suggested that the fetus may be exposed to the active-thiopurine metabolite, 6-thioguaninetriphosphate, in the uterus. A recent study using knock-in mice harboring the p.Arg138Cys mutation which corresponds to human p.Arg139Cys showed that oral administration of 6-MP at clinical dose induces a severe toxic effect on the fetus harboring the homozygous or heterozygous risk allele. This suggests that NUDT15 genotyping may be required in both women with IBD who are planning pregnancy (or pregnant) and their partner to avoid adverse outcomes for their infant. The risk to the fetus due to maternal thiopurine use is minimal but there are some concerns that are yet to be clarified. In particular, a pharmacogenomic approach to the fetus is considered necessary.

Impact of the coronavirus infectious disease (COVID-19) pandemic on the provision of inflammatory bowel disease (IBD) antenatal care and outcomes of pregnancies in women with IBD.

BACKGROUND: The impact of COVID-19 on pregnant inflammatory bowel disease (IBD) patients is currently unknown. Reconfiguration of services during the pandemic may negatively affect medical and obstetric care. We aimed to examine the impacts on IBD antenatal care and pregnancy outcomes. METHODS: Retrospective data were recorded in consecutive patients attending for IBD antenatal care including outpatient appointments, infusion unit visits and advice line encounters. RESULTS: We included 244 pregnant women with IBD, of which 75 (30.7%) were on biologics in whom the treatment was stopped in 29.3% at a median 28 weeks gestation. In addition, 9% of patients were on corticosteroids and 21.5% continued on thiopurines. The care provided during 460 patient encounters was not affected by the pandemic in 94.1% but 68.2% were performed via telephone (compared with 3% prepandemic practice; p<0.0001). One-hundred-ten women delivered 111 alive babies (mean 38.2 weeks gestation, mean birth weight 3324 g) with 12 (11.0%) giving birth before week 37. Birth occurred by vaginal delivery in 72 (56.4%) and by caesarean section in 48 (43.6%) cases. Thirty-three were elective (12 for IBD indications) and 15 emergency caesarean sections. Breast feeding rates were low (38.6%). Among 244 pregnant women with IBD, 1 suspected COVID-19 infection was recorded. CONCLUSION: IBD antenatal care adjustments during the COVID-19 pandemic have not negatively affected patient care. Despite high levels of immunosuppression, only a single COVID-19 infection occurred. Adverse pregnancy outcomes were infrequent.

Oxypurinol - A novel marker for wastewater contamination of the aquatic environment.

The anti-gout agent allopurinol is one of the most prescribed pharmaceuticals in Germany and is widely metabolized into oxypurinol (80%) as well as the corresponding riboside conjugates (10%) within the human body. To investigate the occurrence of allopurinol and oxypurinol in the urban water cycle an analytical method was developed based on solid phase extraction (SPE) and subsequent liquid chromatography electrospray-ionization tandem mass spectrometry (LC-MS/MS). In raw wastewater concentration levels of oxypurinol ranged up to 26.6 µg L(-1), whereas allopurinol was not detected at all. In wastewater treatment plant (WWTP) effluents, concentrations of allopurinol were

Temporal changes in vascular reactivity in early diabetes mellitus in rats: role of changes in endothelial factors and in phosphodiesterase activity.

The aims of this study were to study the influence of the duration of diabetes, the role of endothelial-derived vasodilators, and the role of phosphodiesterase (PDE) isoform activity in the early changes in vascular reactivity of aortic rings from diabetic rats. Diabetes mellitus was induced in female rats by intravenous streptozotocin (85 mg/kg). Two or 4 wk later, thoracic aortic rings from control and diabetic rats were isolated, and vascular responses to acetylcholine (ACh), S-nitroso-N-acetylpenicillamine (SNAP) [nitric oxide (NO) donor], DMPPO (PDE5 inhibitor), and phenylephrine (PE) were obtained in the presence and absence of endothelium or other drugs. PDE isoform activity was also measured. At 2 wk, responses to ACh and DMPPO were enhanced, whereas those to PE were attenuated in diabetic rats relative to controls. Indomethacin and SQ-29548 (a thromboxane A(2) receptor antagonist), but not N(G)-nitro-L-arginine methyl ester, corrected these differences. The responses to SNAP, and cAMP and cGMP hydrolytic activities, were similar in the two groups. In contrast, at 4 wk, ACh, DMPPO, and PE produced similar responses in the two groups: N(G)-nitro-L-arginine methyl ester rendered the response to PE lower in the diabetic group, and this was corrected by indomethacin, but not SQ-29548, treatment. The response to SNAP was greater in the diabetic group, and this was corrected by DMPPO. Activity of all PDEs was decreased at 4 wk. We conclude that, at 2 wk, there is modulation of thromboxane A(2) production, but no change in the NO system or PDE isoform activities. At 4 wk, a reduction in NO activity is superimposed; at this stage, PDE activity is reduced, together with increased production of vasodilating prostaglandins, possibly as a compensatory mechanism to maintain normal vascular reactivity.

Symmetrically and unsymmetrically bridged methylenebis(allopurinols): synthesis of dimeric potential anti-gout drugs.

Liquid-liquid phase transfer alkylation of 4-methoxy-pyrazolo[3,4-d]-pyrimidine (1a) with a dichloromethane/dibromomethane mixture (3:1, v/v) gave the regioisomeric methylenebis(heterocycles) 3a-5a. These were converted by dilute aqueous sodium hydroxide containing dimethylsulfoxide (DMSO) at concentrations between 0 and 60 vol-% into the methylenebis(allopurinols) 3b-5b by nucleophilic SNAr reactions at C(4). The effect of DMSO on the reaction kinetics was investigated.

Effect of phosphodiesterase 5 inhibitor on alteration in vascular smooth muscle sensitivity and renal function in rats with liver cirrhosis.

Previous studies suggested that increased activity of phosphodiesterase (PDE)5 in the kidneys of cirrhotic rats contributes to sodium retention. This study examined the role of PDE5 in the changes in vascular reactivity, hemodynamics, and sodium excretion in rats with liver cirrhosis. Four weeks after bile duct ligation (BDL) or sham operation (SO), in vitro reactivity of aortic rings to various agents and in vivo effects of a PDE5-selective inhibitor [1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazolo[3,4d]-pyrimidin-4-(5H)-one, DMPPO] were studied. The vasodilator responses to nitroglycerin and S-nitroso-N-acetyl-penicillamine (SNAP) in phenylephrine-precontracted rings without endothelium were attenuated in BDL compared with SO rats. Pretreatment with DMPPO (0.1 microM) enhanced these responses and eliminated the differences between the two groups. Vasodilation to DMPPO itself was also less in BDL rats. The responses to phenylephrine were attenuated in endothelium-rich aorta from BDL relative to SO rats, but they were similar in endothelium-denuded aorta and remained similar despite preincubation with SNAP (0.1 microM) alone or with SNAP and DMPPO. In vivo, BDL rats were vasodilated relative to SO rats; DMPPO (5 mg/kg i.v.) decreased arterial pressure and vascular resistance in both groups equally and caused significant increase in sodium excretion in BDL rats only. In conclusion, the results are in accordance with a possible increase in PDE5 activity in aorta and kidney of cirrhotic rats that results in reduced responses to NO donors and contributes to the increase in sodium retention. PDE5 inhibitors may ameliorate sodium retention in cirrhosis but may worsen vasodilation. Examining the effect of PDE5 inhibitors after chronic administration will be more revealing.

Solid-phase parallel synthesis of 4-beta-D-ribofuranosylpyrazolo[4,3-d]pyrimidine nucleosides.

The synthesis of pyrazolo[4,3-d]pyrimidine nucleoside library using solid-phase parallel synthesis methodology is described. Glycosylation of the trimethylsilyl (TMS) derivative of 1- and 2-(methyl)-1H and 2H-pyrazolo[4,3-d]pyrimidine-5,7-(4H, 6H)-dione (5) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of TMS triflate provided two novel protected nucleosides 6 and 7. The structures of 6 and 7 were assigned by 1H and 2D NMR experiments. Nucleosides 6 and 7 were then transformed to the key intermediates 12 and 15 respectively. Reaction of 12 and 15 with MMTCl resin in the presence of 2,6-lutidine afforded the necessary scaffolds B and C. Different amines (96) were introduced selectively by nucleophilic substitution on scaffolds B and C using solid-phase parallel semi-automated synthesizer. Cleavage of the products from the solid support with 30% HFIP in a parallel fashion yielded nucleoside libraries simultaneously, and they were analyzed and characterized by high-throughput LC-MS.

Determination of drug-protein interactions by microdialysis coupled with liquid chromatography and electrochemical detection based on a nickel hexacyanoferrate modified electrode.

Microdialysis sampling coupled with liquid chromatography-electrochemical detection (LC-ED) was developed and applied to determine the interaction of thiopurine (TP) with bovine serum albumin (BSA). A nickel hexacyanoferrate (NiHCNFe) modified electrode was fabricated and used as the working electrode in LC-ED for the determination of TP. Cyclic voltammetric experiments demonstrate that this chemically modified electrode (CME) can effectively catalyze the electrooxidation of TP. The mechanism of the catalytic oxidation of TP at the CME involves an 'EC' process. In the LC-ED, the CME also shows good stability and reproducibility for the determination of TP. The limit of detection is 5.0 x 10(-7) mol l(-1) for 6-TP with a signal-to-noise ratio of 3. The utility of microdialysis as a quantitative sampling technique for in vitro studies of drug-protein interactions was studied. The microdialysis experiments were performed in phosphate buffer solution (pH 7.4) containing different molar ratios of the drug and protein at 37 degrees C. The collected microdialysis sample with unbound TP was analyzed at the NiHCNFe CME in the LC-ED. The relative recovery of TP determined in vitro is 18.3% at a perfusion rate of 1.0 microl min(-1) and the RSD is about 2.1%. The association constant and the number of binding sites on a BSA molecule calculated with the Scatchard equation are 3.72 x 10(-3) (1 mol(-1)) and 1.51, respectively. This method provides a fast, sensitive and simple technique for the study of drug-protein interactions.

Increased cGMP phosphodiesterase activity mediates renal resistance to ANP in rats with bile duct ligation.

BACKGROUND: Liver disease resulting from common bile duct ligation (CBDL) causes abnormal sodium metabolism that is manifested by resistance to the natriuretic action of atrial natriuretic peptide (ANP). This resistance is corrected both in vitro and in vivo by zaprinast, a selective inhibitor of a guanosine cyclic-3'-5'-monophosphate (cGMP)-specific phosphodiesterase (PDE5). Several other PDEs with affinity for cGMP are expressed in kidney and could also be involved in this response. METHODS: We measured cGMP hydrolysis in inner medullary collecting duct (IMCD) cell homogenates from kidneys of sham-operated and CBDL rats and quantitated the amount of PDE5 protein by Western blotting and immunoprecipitation studies. We also characterized ANP responsiveness in vivo of kidneys of anesthetized sham and CBDL rats by measuring sodium excretion before and after volume expansion (VE). RESULTS: Kinetic analysis of PDE5 activity in homogenates of IMCD cells isolated from kidneys of sham-operated rats indicated a Vmax of 85.3 +/- 1.7 versus 157 +/- 2.9 pmol/mg/min from CBDL rats (P < 0.01), without a difference in Km. Enzyme activity was inhibited competitively by 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazol[3,4d]-pyrimidin-4-(5H)-one (DMPPO), a potent and specific inhibitor of PDE5, with an apparent Ki of 4.5 +/- 0.7 and 4.9 +/- 0.7 nmol/L and an IC50 of 6.1 +/- 0.8 and 8.7 +/- 0.7 nmol/L in sham and CBDL rats, respectively (P = NS). DMPPO exhibited very poor inhibitory activity against the calcium-calmodulin-dependent PDE1 in IMCD homogenates from sham rats (Ki 1.3 +/- 0.1 micromol/L and IC50 1.9 +/- 0.2 micromol/L). Western analysis using an antiserum made against bovine lung PDE5 revealed a twofold increase in PDE5 protein in cytosolic extracts from IMCD of CBDL rat kidneys compared with sham-operated controls, and immunoprecipitation studies indicated that the increase in PDE5 protein accounted for the observed increase in cGMP hydrolysis. DMPPO (10 nmol/L) normalized the blunted ANP-dependent cGMP accumulation by IMCD cells from CBDL rats in vitro. Intrarenal infusion of DMPPO (0.5 nmol/min) in CBDL rats corrected both the impaired natriuretic response to VE and the blunted VE-related increase in urinary cGMP excretion from the infused, but not the contralateral kidney. CONCLUSION: These results demonstrate that renal resistance to ANP in CBDL rats is accompanied by heightened activity of PDE5, which is due largely to an increase in PDE5 protein. Other PDEs could contribute only a minor part to the enhanced cGMP hydrolysis observed in kidneys of CBDL rats. This PDE5-dependent ANP resistance may represent an important contributor to the sodium retention of liver disease.

Effect of DMPPO, a phosphodiesterase type 5 inhibitor, on hypoxic pulmonary hypertension in rats.

1. Cyclic guanosine 3'-5'-monophosphate (cyclic GMP) is the second messenger of important physiologically active mediators controlling the pulmonary vascular tone. To potentiate the effects of cyclic GMP on the pulmonary vasculature, we used DMPPO, a new selective PDE-5 inhibitor, and examined its action in a rat model of hypoxic pulmonary hypertension. 2. Levels of cyclic GMP measured during baseline conditions at 5 and 60 min of perfusion were similar in the perfusate of isolated lungs from normoxic and chronically hypoxic rats and did not differ with time. Pretreatment with DMPPO (1 microM) induced a larger increase in cyclic GMP concentration in the perfusate from chronically hypoxic rat lungs (31+/-36 at 5 min to 1821+/-83 pmol ml(-1) at 60 min) than in normoxic rat lungs (329+/-20 to 1281+/-127 pmol ml(-1), P<0.05). 3. In isolated lungs preconstricted with U-46619, pretreatment with DMPPO (1 microM) potentiated the vasodilator effects of atrial natriuretic peptide (100 pM-10 nM) and sodium nitroprusside (1 pM 10 nM), but did not alter vasodilation to isoproterenol. 4. In conscious rats previously exposed to 15 days hypoxia and studied under 10% O2, DMPPO (0.01, 0.05 and 0.1 mg kg(-1), i.v. bolus) caused a dose-dependent decrease in pulmonary arterial pressure (Pap) with no change in systemic artery pressure (Sap) and cardiac output. 5. Continuous infusion of DMPPO (0.1 mg kg(-1) h(-1) i.v. by osmotic pumps) in rats exposed to 10% O2 during 2-weeks reduced the Pap (P<0.05) and the degree of muscularization of pulmonary vessels at the alveolar wall (P<0.01) and alveolar duct levels (P<0.05) despite no significant change in right ventricular hypertrophy. 6. These results suggest that cyclic GMP phosphodiesterase inhibition may selectively dilate pulmonary circulation during chronic hypoxia.

Identification of a transcription factor like protein at the TOR locus in Leishmania mexicana amazonensis.

The TOR gene (TOxic nucleoside Resistance gene) was mapped to a 2.3 kb fragment on the amplified DNA from tubercidin resistant Leishmania (TUB). This DNA fragment conferred upon wild type cells resistance to tubercidin, inosine dialdehyde, formycin A and B and allopurinol riboside and a reduced ability to accumulate purine nucleobases and nucleosides. These properties were characteristic of the parental TUB cells which carried the intact amplified DNA and have been hypothesized to be caused by a reduction in the activity of the multiple purine transporters within this organism. The TOR gene was found to be partially homologous to the rodent and human Oct-6/SCIP/Tst-1 gene. It lacked, however, the POU specific domain of this class of transcription factors and contained only the first two helices of the POU homeodomain. This truncated homeodomain was not required to confer resistance upon wild type cells to toxic nucleosides, suggesting that TOR was not a repressor with independent DNA binding capability.

Effects of cyclic GMP elevation on isoprenaline-induced increase in cyclic AMP and relaxation in rat aortic smooth muscle: role of phosphodiesterase 3.

1. In rat aortic rings precontracted with phenylephrine, the beta-adrenoceptor agonist isoprenaline (10 nM to 30 microM) produces greater relaxant effects in preparations with endothelium than in endothelium-denuded preparations. The aim of this study was to determine the mechanisms involved in this effect and in particular investigate the possibility of a synergistic action between adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP). 2. Isoprenaline-induced relaxation of rat aortic rings precontracted with phenylephrine was greatly reduced by the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 300 microM) or the soluble guanylate cyclase inhibitors methylene blue (10 microM) or IH-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) but unaffected by indomethacin (10 microM), a cyclo-oxygenase inhibitor. Similarly, in intact rings, the concentration-response curve of forskolin (10 nM to 1 microM) was shifted to the right upon endothelium removal or treatment with methylene blue. 3. In endothelium-denuded rat aortic rings, isoprenaline-induced relaxation was potentiated by the guanylate cyclase activators atrial natriuretic factor (ANF, 1 to 10 nM) and sodium nitroprusside (SNP, 1 to 10 nM), and to a greater extent in the presence of the cyclic GMP-specific phosphodiesterase (PDE 5) inhibitor, 1,3dimethyl-6-(2-propoxy-5-methane sulphonylamidophenyl) pyrazolo [3,4-d] pyrimidin-4-(5H)-one (DMPPO, 30 nM). Relaxation induced by isoprenaline was also potentiated by the cyclic GMP-inhibited PDE (PDE 3) inhibitor cilostamide (100 nM). 4. Intracellular cyclic nucleotide levels were measured either in rat cultured aortic smooth muscle cells or in de-endothelialized aortic rings. In both types of preparation, isoprenaline (5 nM and 10 microM) increased cyclic AMP levels and this effect was potentiated by cilostamide (10 microM), by rolipram, a cyclic AMP-specific PDE (PDE 4) inhibitor (10 microM) and by cyclic GMP-elevating agents (50 nM ANF or 30 nM SNP plus 100 nM DMPPO). In isoprenaline-stimulated conditions, the increase in cyclic AMP induced by rolipram was further potentiated by cilostamide and by cyclic GMP-elevating agents. Cilostamide and cyclic GMP-elevating agents did not potentiate each other, suggesting a similar mechanism of action. 5. We conclude that in vascular smooth muscle (VSM) cells an increase in cyclic GMP levels may inhibit PDE 3 and, thereby, cyclic AMP catabolism. Under physiological conditions of constitutive NO release, and to a greater extent in the presence of the PDE 5 inhibitor DMPPO, cyclic GMP should act synergistically with adenylate cyclase activators to relax VSM.

Cardiovascular effects of a novel, potent and selective phosphodiesterase 5 inhibitor, DMPPO: in vitro and in vivo characterization.

1. The aim of this study was to investigate the cardiovascular effects of a novel, potent and specific phosphodiesterase 5 (PDE 5) inhibitor, 1,3 dimethyl-6-(2-propoxy-5-methane sulphonylamidophenyl)-pyrazolo[3,4-d]pyrimidin-4-(5H)-one (DMPPO) in phenylephrine-precontracted rat aortic rings and different in vivo rat preparations. 2. DMPPO elicited a concentration-dependent relaxation of rat aortic rings with functional endothelium. DMPPO-induced relaxation was abolished by endothelium removal or pretreatment with the soluble guanylate cyclase inhibitor, methylene blue (10 microM). 3. In aortic rings without endothelium, the potency (pD2= -log10 EC50) of atrial natriuretic peptide (ANP) to induce relaxation increased from 8.13 +/- 0.05 in the absence of DMPPO, to 8.32 +/- 0.05 and 8.52 +/- 0.08 in the presence of 30 nM and 100 nM DMPPO, respectively. Similarly, the potency of sodium nitroprusside (SNP) in inducing relaxation increased from 7.38 +/- 0.07 in the absence of the PDE 5 inhibitor to 8.07 +/- 0.11 and 8.15 +/- 0.08 in the presence of 30 nM and 100 nM DMPPO, respectively. In contrast, relaxation to the adenylate cyclase activator, forskolin, was unchanged by DMPPO (100 nM). 4. In rings without endothelium, DMPPO (100 nM) increased by 2.5 fold intracellular levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP). Moreover, DMPPO (100 nM) potentiated the increases in cyclic GMP levels induced by ANP (30 nM) by 3 fold and SNP (30 nM) by 2.7 fold. Adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were not modified by DMPPO. 5. In anaesthetized normotensive or spontaneously hypertensive rats (SHR), DMPPO (2 and 5 mg kg-1, i.v.) lowered blood pressure without affecting heart rate. Similarly, in conscious SHR, orally administered DMPPO (5 mg kg-1) induced a 25 mmHg decrease in blood pressure for at least 7 h without modifying heart rate. Meanwhile, urinary cyclic GMP was increased by 50% whereas cyclic AMP remained unchanged. 6. In normotensive anaesthetized rats, sodium nitroprusside (SNP) (i.v. bolus) induced a decrease in blood pressure which rapidly returned to baseline. In DMPPO (1 mg kg-1, i.v.)-treated rats, the hypotensive effects of SNP (10 to 100 micrograms kg-1) were prolonged over time whereas the peak effect was unchanged. 7. In pithed rats, phenylephrine (i.v. bolus) induced dose-dependent increases in blood pressure. Pretreatment with DMPPO (5 mg kg-1, i.v.) partially inhibited the pressor response to phenylephrine (0.3 to 100 micrograms kg-1). 8. In conclusion, the potent and selective PDE 5 inhibitor, DMPPO, produces relaxation in isolated vessels in the presence of a cyclic GMP drive and reduces blood pressure of intact animals. Its high oral bioavailability and long duration of action should make it a useful tool to study the role of cyclic GMP in various biological systems.

Characterization of a novel potent and specific inhibitor of type V phosphodiesterase.

Guanosine cyclic 3':5'-monophosphate (cGMP) plays a crucial role in regulating vascular smooth muscle contractile state. In rat aortic smooth muscle cells (RSMC) three isozymes of phosphodiesterase (PDE) may be involved in the degradation of cGMP, namely PDE I, PDE III, and PDE V. To study the effective contribution of PDE V to the control of intracellular cGMP levels, a specific and potent PDE V inhibitor 1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazolo[3, 4d]- pyrimidin-4-(5H)-one (DMPPO) was synthesized. DMPPO is a competitive inhibitor with respect to cGMP (Ki = 3 nM) and displayed high selectivity for PDE V as compared to other PDE isozymes. DMPPO strongly potentiated the cGMP response of atrial natriuretic peptide- or sodium nitroprusside-treated RSMC (EC50 = 0.5 microM). In addition, similar intracellular cGMP levels were obtained in the presence of a saturating concentration of DMPPO or 3-isobutyl-1-methylxanthine, a nonspecific PDE inhibitor, suggesting that cGMP is almost exclusively hydrolyzed by PDE V in RSMC. Stimulation of RSMC with atrial natriuretic factor resulted in accumulation of cGMP in the extracellular media. This egression was shown to be proportional to the intracellular level of cGMP and a first-order rate constant of 0.04 min-1 was determined for the egression process. DMPPO did not interfere with the efflux and allowed us to show that intracellular cGMP levels are mainly controlled by PDE V, rather than by egression in RSMC. DMPPO is, therefore, a useful tool for determining the role of PDE V in the control of cGMP levels in living cells and tissues.

Effects of probenecid on the pharmacokinetics of allopurinol riboside.

Allopurinol riboside is an experimental agent for the treatment of leishmaniasis and American trypanosomiasis. Previous studies showed that after oral administration, unexpectedly low levels of allopurinol riboside in plasma are attributable to incomplete absorption and rapid renal clearance. In this randomized, crossover evaluation in healthy volunteers, probenecid reduces the renal clearance of allopurinol riboside, extends the half-life of allopurinol riboside in plasma, and triples the levels of allopurinol riboside in plasma.

Inhibition by 2,6-dithiopurine and thiopurinol of binding of a benzo(a)pyrene diol epoxide to DNA in mouse epidermis and of the initiation phase of two-stage tumorigenesis.

The chemotherapeutic agent 6-mercaptopurine was previously shown to inhibit the binding of 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydro-benzo(a) pyrene (BPDE-I) to DNA in Chinese hamster ovary cells. Two compounds related to 6-mercaptopurine, 2,6-dithiopurine (DTP) and thiopurinol (TP), have been tested for inhibition of the binding of BPDE-I to epidermal DNA in mouse skin. Doses of test compound (0.2-20 mumol) or solvent control were applied to the shaved backs of female SENCAR mice. Fifteen min later, 200 nmol [3H]BPDE-I were applied to the same area and 3 h later the mice were sacrificed and epidermal DNA was purified and adduct formation was quantitated radiometrically. At the highest doses studied, DTP and TP inhibited DNA binding by 90 and greater than 80%, respectively. The dose necessary to inhibit DNA binding by 50% was about 0.8 mumol for DTP and about 2 mumol for TP. To test whether this protective effect was long-lasting, the time between application of purinethiol and [3H]BPDE-I was systematically increased. Although the level of protection was decreased by increasing the time between applications, both compounds inhibited binding 50-60% even after 24-48 h. A radioactive compound tentatively identified as a TP-BPDE-I adduct could be recovered from epidermal homogenates following topical application of TP and BPDE-I. We used a standard two-stage initiation-promotion protocol to test the effects of these compounds on mouse skin carcinogenesis. Mice were treated with 0, 1, or 10 mumol of either TP or DTP, and 15 min later were treated with an initiating dose of BPDE-I (200 nmol). Twice weekly promotion with 12-O-tetradecanoylphorbol-13-acetate was begun 2 weeks later and continued for 23 weeks. A dose-dependent inhibition of tumor incidence and multiplicity was noted with both compounds. Treatment of skin with 10 mumol of DTP prior to initiation lowered the number of papillomas per mouse by greater than 90% compared to solvent controls; a 10-fold lower dose resulted in about 50% inhibition. The 10-mumol dose of TP resulted in about 50% inhibition. Mice were examined for 50 weeks for the presence of squamous cell carcinomas. Compared to the positive control group, 10 mumol DTP inhibited carcinoma incidence and lowered the total number of carcinomas by 90-95%. Treatment with 10 mumol TP had no significant effect on carcinoma incidence, and only slightly lowered the total number of carcinomas.

Placebo controlled treatment of Ecuadorian cutaneous leishmaniasis.

Pentavalent antimony has been considered to be the standard treatment for leishmaniasis, but more recently, the orally administrable agent allopurinol ribonucleoside has been the subject of several clinical trials. In this study, these two agents were evaluated in patients with Ecuadorian cutaneous leishmaniasis. Patients were randomly assigned to the two treatment groups. The mean reduction in lesion size for the 28 patients treated with Pentostam (20 mg Sb/kg/day intramuscularly for 20 days) was 61%, 23%, and 11% after one, two, and three weeks, respectively. There was a wide range in the individual values, and some lesions markedly enlarged in the first week of therapy. An initially healed lesion was defined as one that had greater than 80% re-epithelialized by the 1.5-month post-treatment followup. All Pentostam patients demonstrated this degree of lesion resolution (100% initial healing rate), but one patient showed evidence of relapse at the three month followup resulting in a 96% complete healing rate for the 12 month observation period. Patients in the untreated control group demonstrated a strikingly high rate of healing with 9 of 12 patients having re-epithelialized all lesions after 1.5 months observation (75% initial healing rate). The mean reduction in lesion size for the untreated patients was 56%, 29%, and 25% after one, two, and three weeks, respectively. Twenty-one patients received allopurinol ribonucleoside (1,500 mg QID) plus probenecid (500 mg QID) for 28 days. Lesions in nine of these patients were healed at the time of the 1.5 month followup (41% healing rate).(ABSTRACT TRUNCATED AT 250 WORDS)